New Humanized Antibody Breaks Sepsis Inflammation Cycle at Its Source

Sepsis kills millions annually by triggering a dysregulated immune response that leads to cytokine storms, organ failure, and death. A central but underexplored driver is citrullinated histone H3 (CitH3), a modified nuclear protein released when neutrophils form extracellular traps (NETosis). Elevated CitH3 perpetuates inflammation by amplifying both NETosis and macrophage pyroptosis, yet no clinically viable antibody-based therapy had been designed to neutralize it broadly.

This study describes the systematic development of a humanized anti-CitH3 monoclonal antibody (hCitH3-mAb) engineered from an existing murine precursor using AI-assisted antibody humanization. The antibody was optimized for high affinity and specificity across citrullinated residues at positions R2, R8, R17, and R26 of histone H3—sites targeted by both PAD2 and PAD4 enzymes—and was manufactured at scale in CHO cells with >99.5% purity. Binding kinetics confirmed nanomolar affinity, validating the humanization strategy.

In murine sepsis models using LPS challenge and live Pseudomonas aeruginosa infection, hCitH3-mAb significantly reduced pro-inflammatory cytokine production, lowered mortality rates, and attenuated acute lung injury. Importantly, the antibody enhanced bacterial phagocytosis in the lungs, spleen, and liver, suggesting it restores rather than globally suppresses immune function. A novel pre-equilibrium digital ELISA (PEdELISA) platform was used to detect ultra-low CitH3 concentrations in plasma, enabling identification of an optimal therapeutic window for antibody intervention.

A key mechanistic discovery is the CitH3–PAD2 feedback loop. CitH3 released extracellularly activates Toll-like receptor 2 (TLR2) on macrophages, triggering intracellular calcium release. This calcium surge drives PAD2 auto-citrullination and its translocation from the cytosol into the nucleus, where it citrullinates histone H3 to produce more CitH3—completing a vicious amplification cycle. The hCitH3-mAb interrupts this loop at the extracellular step, effectively preventing downstream TLR2 activation and restoring macrophage homeostasis.

These findings establish hCitH3-mAb as both a mechanistic tool and a promising therapeutic candidate for sepsis and potentially other immune-mediated inflammatory diseases. Caveats include the preclinical nature of the data and the need for human clinical validation, particularly regarding optimal dosing windows and off-target effects in diverse patient populations.